Anxiety disorders are the most prevalent class of neuropsychiatric illness worldwide. These disorders, along with their comorbidities with a wide range of medical and psychiatric illnesses, cause significant functional impairment. Recent studies in animal models suggest that bacterial colonization of the intestine is critical to the normal development of many aspects of brain function, including learning and memory and emotional processes such as anxiety. It is known that colonization of gut microbiota in germ-free (GF) mice normalizes anxiety-like behavior, and that brain chemistry and behavioral responses are altered when gut microbiota is manipulated. However, how the indigenous gut microbiota regulate brain serotonergic system and anxiety behavior is poorly understood. Our central hypothesis is that dietary tryptophan intake directly affects serum tryptophan levels, which alters central levels of 5- hydroxytryptamine (5-HT, serotonin) in the absence of gut microbiota, and that gut microbiota modulate host behavior by regulating the amount of tryptophan available to the central nervous system. Our hypothesis is formulated on the basis of our preliminary data that colonization of gut microbiota in GF mice decreases serum tryptophan concentration by ~60%, indicating that the higher serum tryptophan level in GF mice is due to the absence of gut microbiota, and that colonization of GF mice with gut microbiota increases anxiety-like behavior. The rationale for the proposed research is that once the role of gut microbiota in central serotonergic signaling and behavior is well understood, it may be possible to develop new therapeutics by targeting the metabolic activities of the gut microbiota. This application will clarify the role of dietary tryptophan and bacterial tryptophanase in regulating central serotonin and anxiety using a gnotobiotic mouse model in two Specific Aims: Aim 1) How does dietary tryptophan regulate brain serotonergic system and anxiety through indigenous gut microbiota? Aim 2) What is the role of bacterial tryptophanase in central serotonergic signaling? Our approach is innovative because we will perform direct experimental manipulations of dietary tryptophan and gut bacteria in gnotobiotic mice, with simultaneous measurements of plasma tryptophan, brain serotonergic function, and anxiety-like behavior. The proposed research is significant because it will define the role of dietary tryptophan and gut bacterial tryptophanase i regulating central serotonin. This knowledge will advance our understanding of the role of microbial tryptophanase in modulating brain function and behavior, which may lead to novel approaches such as new druggable microbial targets for the treatment of a variety of mental illnesses.